Phenotypic Classification Of Gastric Signet Ring Cell Carcinoma And Its Relationship With K-ras Mutation And Prognosis

Objective:1) To elucidate the characteristics and distribution of mucus in SRCC and to investigate the mechanism of heterogeneity in mucin expression of tumor cells.2) To investigate phenotypic classification of SRCC and its relationship with K-ras mutation and prognosis, estimate feasibility of K-ras gene mutation as target for molecular targeted therapy (MTT) and provide a theoretical basis to therapy and prognostic evaluation.Methods:1) A total of 163 patients with gastric SRCC who underwent curative surgery from 2000 to 2008 were followed up by telephone in this study.2) Mucin histochemical method was performed in 163 cases of SRCC and 30 cases of normal mucosa. Meanwhile, Immunohistochemistry was performed with MUC1, MUC5AC, MUC6 antibodies as gastric phenotypic markers and MUC2, CDX2 antibodies as intestinal phenotypic markers, according to the expression of phenotypic markers, tumors were classified into three different types:gastric type, intestinal type and mixed type.3) Genomic DNA was extracted by standard method, K-ras gene was amplified by polymerase chain reaction (PCR) and analyzed mutation in codon12,13 and 61 by DNA direct sequencing.Results:1) Clinical follow-up were available for 143 patients (87.7%),62 of them were alive, and 81 of them were die.2) Histopathological findings:cases of typeⅠSRCC were 137, while cases of typeⅢSRCC were 26.3) The cases containing neutral mucus, sialomucin or sulfomucin in carcinoma and normal mucosa were 69.9%(114/163),42.3% (69/163),19%(31/163) and 100%(30/30),0%(0/30),0%(0/30) respectively. The expression rates of MUC1, MUC5AC, MUC6, MUC2 and CDX2 in 163 cases of SRCC and 30 cases of normal gastric mucosa were 20.9%(34/163),73.6%(120/163), 28.8%(47/163),46.6%(76/163),39.9%(64/163) and 50%(15/30),80%(24/30),60%(18/30), 0%(0/30),0%(0/30) respectively.4) The cases containing neutral mucus in carcinoma was significantly lower than that in normal mucosa (P＜0.05). Inversely, the cases containing sialomucin or sulfomucin in carcinoma were significantly higher than that in normal mucosa (P＜0.05).5) The positive rates of MUC1 and MUC6 in carcinoma were significantly lower than that in normal mucosa (P＜0.05). Inversely, the rates of MUC2 and CDX2 expression in carcinoma were significantly higher than that in mucosal carcinoma (P＜0.01 and P＜0.05). The cases containing neutral mucus in intramucosal carcinoma, carcinoma with tumor size≤5.0cm, carcinoma without lymph node metastasis and with (Ⅰ+Ⅱ) stages were more than that in submucosal carcinoma, carcinoma with tumor size＞5.0cm, carcinoma with lymph node metastasis and (Ⅲ+Ⅳ) stages (P＜0.05). Inversely, the cases containing sialomucin or sulfomucin in intramucosal carcinoma were less than that in submucoasl carcinoma (P＜0.05). The cases containing sialomucin or sulfomucin in carcinoma with lymph node metastasis and (Ⅲ+Ⅳ) stages were significantly more than that without lymph node metastasis and (Ⅰ+Ⅱ) stages (P＜0.05).6) There was no relationship between phenotypic markers expression and morphological classification of SRCC.7) The expression rate of MUC2 was positive related to depth of invasion and lymph node metastasis (P=0.001 and P=0.002). The expression rate of CDX2 was positive related to tumor size, depth of invasion and TNM stage (P=0.004 and P=0.001). The expression rate of MUC5AC was negative related to depth of invasion (P=0.001).8) According to the expression of phenotypic markers,63 cases (38.6%) were gastric type,71 cases (43.5%) were mixed type,29 cases (17.9%) were intestinal type.9) More cases of intestinal type of SRCC had tumor size＞5.0cm, wall invasion deeper than submucosa layer, lymph node metastasis and (Ⅲ+Ⅳ) stage (P＜0.01) than other types.10) The frequency of K-ras gene mutation was 12.27%(20/163), all mutational loci were found in codon 12. Among the 20 cases of mutation,8 cases showed mutation of GGT→GTT,10 cases showed mutation of GGT→GAT,1 case showed mutation of GGT→AGT,1 case showed mutation of GGT→TGT. No mutation at codon 13,61 was deteted.11) More K-ras gene mutation was found in cases with MUC5AC (-), MUC6 (-) and intestinal type of SRCC.12) Survival analysis showed that median survival period of cases with MUC2 (+) was shorter than cases with MUC2 (-) (P＜0.05). Intestinal and mixed types were associated with shortened median survival period (P＜0.05).13) Univariate survival analysis showed that depth of invasion and MUC2 expression were prognostic factors, while multivariate survival analysis showed that depth of invasion and MUC2 expression were independent prognostic factors (P＜0.05).Conclusions:1) Phenotypic classification was correlated with biological behaviors and prognosis in SRCC.2) The frequency of K-ras gene mutation was 12.27%, all mutational loci were found in codon 12.3) K-ras gene mutation was more common in intestinal type, indicated that it may play an important role in phenotypic differentiation.4) There had different genetic pathways according to the phenotypic marker expression patterns and it might play a role in the tumorigenesis of the SRCC.